Method for blood film preparation

ABSTRACT

Sample preparation apparatus for forming a thin layer of diluted blood specimen on a substrate by centrifugal force includes a sample probe having coaxial blood and diluent conduits coupled to respective reversibly drivable peristaltic pumps having forward operation intakes connected to a diluent supply. A one-way clutch limits the diluent pump to forward operation and the pumps are geared to produce a blood-to-diluent displacement ratio of 2:1. A substrate holder is mounted on a carriage for selective insertion into and removal from a sealed centrifuge chamber in which it is spun at a high angular rate by a drive motor. Means are provided to form a curtain of flowing liquid within the chamber surrounding the spinning substrate to collect spun-off blood particles and aerosol. The liquid forming the curtain is drained by an exhaust pump which operates after spinning has ceased so as to create a partial vacuum for draining off residual aerosol prior to unsealing the centrifuge chamber.

This is a division of application Ser. No. 669,015, filed Mar. 22, 1976,now Pat. No. 4,016,828.

BACKGROUND OF THE INVENTION

This invention relates generally to a clinical method for medicaldiagnosis and, more particularly, to an improved method for thepreparation of blood sample films or "smears" on microscope slides orsimilar substrates. While applicable to all techniques for evaluation ofthe resulting smears, the method and apparatus are especially adaptedfor use in connection with automated blood cell analysis usingcomputerized pattern recognition systems.

The traditional method of preparing blood smears on microscope slidesfor evaluation by physicians or medical technicians consists of placinga small quantity of the blood specimen on a microscope slide and thenmanually wiping the blood across the slide with the edge of anotherslide or a cover glass. While reasonably satisfactory for thepreparation of smears for microscopic examination by skilled techniciansor physicians, the obvious variations in results from slide to slideinherent in such a manual technique obviously renders it whollyunsuitable to preparation of slides for evaluation by automated patternrecognition apparatus.

An early attempt to achieve a high degree of control and uniformity inthe preparation of blood smears is described in U.S. Pat. No. 3,577,267granted to K. Preston, Jr. et al. on May 4, 1971. The method andapparatus proposed by Preston et al. involves placing a small specimenon a substrate and then spinning the substrate at high speeds, thusutilizing centrifugal force to form an evenly distributed thin layer ofblood on the substrate surface with a minimum of disturbance to bloodcell morphology. This involves rapid acceleration of the substrate andspinning at high speeds in the range of from 4,000 to 10,000 rpmaccording to the Preston et al patent. Consequently, the greater portionof the original specimen is spun off the slide, much of it in the formof aerosol. A shield is provided around the spinner to preventwidespread scattering of the spun-off blood but does little or nothingto prevent aerosol from entering the atmosphere of the laboratory.

Inasmuch as some of the blood specimens routinely handled in hospitaland private clinical laboratories contain pathogenic organisms, the useof spinners and the resultant dispersal of aerosol constitutes a healthhazard for clinical personnel, exposure to hepatitis virus being aparticular problem.

The containment of aerosol by disposing the spinner in an enclosedchamber such as that shown in U.S. Pat. No. 3,705,048 to J.J.J.Staunton, represents a partial solution only because the aerosolpersists within the chamber and eventually escapes into the atmospherewhen the cover is opened.

Another problem encountered in the utilization of centrifuges to prepareblood smears is the variation in hematocrit from specimen to specimenwhich makes it necessary to adjust the spinning parameters (e.g.,acceleration, speed and spinning time) in order to obtain a degree ofuniformity in the resulting smear. In this connection, reference may behad to U.S. Pat. No. 3,906,890 to Amos et al. describing a blood smearcentrifuge in which spinning time is automatically adjusted inaccordance with the hematocrit of the specimen. Obviously, it would bepreferable if the spinning parameters could be the same for all or atleast the vast majority of blood specimens and, to this end, it has beensuggested (J. W. Bacus, Erythrocyte Morphology and Centrifugal "Spinner"Blood Film Preparations, J. of Histochemistry and Cytochemistry, Vol.22, No. 7, pp. 506-516) that it would be desirable to adjust thehematocrit in order to permit slides to be spun at the same speed andfor the same time. Adjustment of the hematocrit may be accomplished bydilution of the blood specimen with physiological saline. To avoid thenecessity of adding sample dilution to the procedures performed by theoperator and to ensure uniformity in the proportions of sample anddiluent, it is highly desirable that the dilution be carried outautomatically. It is to the automatic accomplishment of dilution as wellas the prevention of atmospheric contamination with aerosol that thepresent invention is addressed. It is the basic general object of theinvention to provide a blood smear preparation method which overcomes ormitigates problems of the prior art as outlined hereinabove.

A more specific object is the provision of a sample preparation methodfor preparing blood smears on substrates which are particularly suitablefor evaluation by computer operated pattern recognition apparatus or thelike which produces smears of high uniformity and reproducibility.

A further object is the provision of a method in accordance with thepreceding objects which effects blood specimen dilution and spinningessentially without operator intervention and which effects theformation of a blood smear by centrifugal action without the attendanthazard of dispersing possibly pathogenic blood particles and aerosolinto the atmosphere.

SUMMARY OF THE INVENTION

To the accomplishment of the foregoing objects and the attainment offurther objects and advantages which will become apparent as thisdescription proceeds, the invention contemplates a method of blood smearpreparation in which a blood specimen is deposited on a planar substratewhich is then spun at a high angular rate about an axis normal to itsplane while the substrate is surrounded by a flowing curtain of liquidpositioned to intercept and collect blood particles and aerosols spunoff the substrate.

The invention is embodied in apparatus consisting of two basiccomponents: (1) a sample aspirating and diluting probe which picks up aspecimen quantity of blood, automatically mixes it with diluent in afixed proportion, and then, on signal, discharges the diluted specimenand (2) a substrate spinner or centrifuge which, when operative, iscompletely enclosed in a sealed housing to prevent contamination of thesurrounding area by blood particles or aerosol generated as a result ofcentrifuge operation. Means are provided to form a curtain of flowingliquid within the enclosure surrounding the substrate while spinning tointercept and collect blood particles and aerosol. The liquid formingthe curtain is then directed to a drain. Discharge of thecurtain-forming liquid to drain is accomplished by positive exhaustpumping which is continued after rotation of the centrifuge has ceasedin order to create a partial vacuum within the enclosure and thus ensurethe removal of residual aerosol prior to opening of the enclosure.

In accordance with particular additional features of the invention, thecentrifuge includes a substrate holder mounted on a carriage which maybe withdrawn or retracted from the enclosure to permit insertion of asubstrate. Advancing the carriage into the housing automatically effectslocking of the substrate in the holder and sealing of the centrifugeenclosure; automatically effects coupling of a drive motor to thesubstrate holder; and locks the carriage in the enclosure to prevent itsretraction during the spinning cycle.

The probe is employed by the operator to deposit a metered quantity ofdiluted blood onto the substrate, preferably after it is loaded into thesubstrate holder. The internal surfaces of the probe which have been incontact with the blood specimen are cleansed by discharge of diluentsubsequent to depositing the sample on the substrate and the externalsurfaces cleansed during the operation of the centrifuge by a spray ofliquid from the same source as that forming the liquid curtain.

Through these various features, a safe, efficient, reliable and easy tooperate system is provided for the preparation of blood sample smears.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of the blood film preparation apparatus ofthe present invention;

FIG. 2 is a flow chart of the overall operation of the apparatus of FIG.1;

FIG. 3 is a perspective view of the sample aspirating/diluting probe ofthe apparatus of FIG. 1;

FIG. 4 is a longitudinal sectional view of the tip portion of the probeof FIG. 3;

FIG. 5 is a block diagram of the pumping and control system associatedwith the probe of FIG. 4;

FIG. 6 is block-logic diagram of the control system of FIG. 5;

FIG. 7 is a sectional view of the centrifuge portion of FIG. 1, thesection being taken in the plane of the spin axis;

FIG. 8 is a section on line 8--8 of FIG. 7;

FIG. 9 is a sectional view of the substrate holder portion of theapparatus as shown in FIG. 7, taken on the spin axis;

FIG. 10 is a plumbing diagram for the apparatus of FIG. 1 including theprobe wash system; and

FIG. 11 is a block-logic diagram for the apparatus of FIG. 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring now to the drawings and first in particular to FIG. 1, thereis shown and indicated generally as 11, a blood film preparationapparatus in accordance with the present invention. As alreadymentioned, there are two major components of the apparatus, a centrifugeassembly 13 and a sampling probe 15. Preferably, both are mounted on aself-contained, easily movable cart unit 17. Also appearing in FIG. 1 isa diluent container 19, which supplies diluent (usually physiologicalsaline) to probe 15, as well as associated electrical conductors andliquid conduits described in greater detail hereinbelow. Centrifugeassembly 13 includes a substrate holder 21 on which is disposed asubstrate 23, e.g., a standard microscope slide. Substrate holder 21 ismounted on a carriage 27 which rides on tracks extending into acentrifuge chamber defined by an enclosure 29. A handle member 25 on theouter end of carriage 27 enables the carriage to be advanced intoenclosure 29 or retracted therefrom in a manner which will becomeapparent as this description proceeds. In FIG. 1, substrateholder/carriage assembly 21, 27 is illustrated in the retracted or openposition, i.e., ready to permit insertion or removal of a substrate.

At this juncture, the basic operational sequence of the apparatus willbe related as a preface to, and to facilitate understanding of, thestructural aspects of the invention. The operator, after turning on theapparatus and getting an indication on an indicator 31 that the systemis ready for operation (possibly after purging the system with a rinsingliquid, such as water, by operating a purge button 33) aspirates a bloodspecimen by inserting the tip of probe 15 into the blood sample andpressing a control switch 35 on the handle of the probe. This causesaspiration of a predetermined quantity of the blood sample into theprobe wherein it is mixed with a diluent in a manner to be describedpresently. Thereupon, the operator places the tip of the probe oversubstrate 23 and actuates control switch 35 once again. This causesdischarge of a metered quantity of the diluted blood sample onto thesubstrate. The operator then moves the substrate holder/carriageassembly into chamber 29, by pushing on handle 25, and concomitantlyautomatically initiates operation of the centrifuge. Movement of theholder/carriage into chamber 29 also completes closure of and seals thechamber.

The remainder of the operating cycle can best be appreciated byreference to the flow diagram shown in FIG. 2. From a start block 37 atthe beginning of the program, a signal flows to a decision block 39where a start check is made to determine if the substrateholder/carriage assembly has been moved into the centrifuge chamber. Ifnegative, recycling occurs; if affirmative, indicating that thesubstrate holder/carriage is in the closed position, the substrate islocked in place so that rotation can safely begin as represented byfirst instruction block 41. This condition is visually represented tothe operator by extinguishment of a "ready light."

The next instruction block, 43, represents the turn-on of the fluidsystems associated with the apparatus and more specifically the inletvalve and exhaust or drain pump for the liquid forming the curtainaround the spinning substrate and the pump for the sample diluent. Inthis connection, the liquid forming the curtain is normally water andmay be referred to as such hereinafter. Accordingly, the explanatorylegend beside the block 43 in FIG. 2 refers to the "water solenoid" and"water drain pump."

The next instruction block 45, controls the spin program, viz., theinitiation, speed regulation, and termination of the centrifugeoperation. It should be noted that spinning is not initiated until afterthe water forming the protective curtain has been turned on.

When the spinning cycle is completed and the centrifuge stopped, thewater curtain is turned off by turning off the water solenoid valve and,after a 4 -second delay, exhaust pump operation is terminated asindicated by instruction block 47. Thereafter, the centrifuge motor ispositioned as instructed by block 49 to permit the engagement anddisengagement of coupling between the substrate holder and the motor.Block 51 is entered, the ready reset, rotate set to 0 to lock the motorin position, the lock solenoid opened to permit withdrawal of thesubstrate and the diluter pumps turned off. During the time that thewater valve is open, water is provided not only to the inside of theenclosure 29 where it forms a curtain to collect and carry away spun-offblood particles and aerosol, but is also directed to cleanse theexternal portion of the probe.

FIG. 3 is a perspective view of the probe 15 of FIG. 1. As illustrated,it includes a grip portion 53 and a shank portion 55, the tip of whichis immersed in a blood sample to aspirate a specimen to be used on thesubstrate. A flexible tubular sheath 57, connected to the grip end ofthe probe, houses tubing for the diluent and electrical controlconductors. Adjacent grip 53 is the switch or sequence button 35 whichcontrols the probe functions involved in aspirating blood and expellingit in a diluted condition onto the substrate. Above button 35 is an LEDdisplay 58 for indicating the status of the probe.

In order to achieve satisfactory results in the preparation of bloodspecimen films in accordance with the present invention, it is essentialthat mixing of blood and diluent by the probe be carried out in aneffective manner. A cross-section of the probe construction whichaccomplishes such mixing is shown in FIG. 4 which illustrates a lowersegment of the shank portion 55. While only partially illustrated,substantially the entire shank of the probe is made up of coaxiallydisposed hollow tubular members 59 and 61. The inner member 61, whichdefines a conduit 62 for the aspiration of a blood specimen preferablyis made of stainless steel; the outer member 59 which defines a diluentpassage 60 surrounding the inner member preferably is fabricated ofTeflon or the like.

The lower end of outer member 59 is conically tapered to form a smalldiameter tip 64 which is easily inserted into a blood sample cup toaspirate a specimen and facilitates its subsequent deposit, in dilutedform, on the substrate.

Inner tubular member 61 terminates at a distance from the taperedportion 64 of outer member 59 in communication with a mixing region orchamber there located. The mixing chamber, formed by the coaction ofindividual or integral mechanical elements 63, 66, the physical detailsof which are not material, takes the form of a thin discoid upperportion 68 and an annular lower portion 69 interconnected by a pluralityof axial passages 72. Upper portion 68 of the chamber is in direct flowcommunication with diluent conduit 60 via a plurality of ports 74 andlower chamber portion 69 communicates with an axial duct 64' opening atthe external tip of the probe. A cylindrical projection 63' extends fromelement 63 into the inner end of duct 64' with a small clearance, e.g.,to create an annular flow restriction at that point which aids in themixing of blood and diluent and breaks up any clots which might form.

Conduit 60 is filled with diluent and is in communication with a diluentpump as hereinafter described; similarly, conduit 62, normally alsofilled with diluent, is in communication with a blood sample pump.During aspiration of a blood specimen, only the sample pump is operativeand blood is drawn through tip duct 64' and the mixing chamber into thelower end portion of conduit 62. Thereafter, when the diluted specimenis to be deposited on the substrate, both the diluent and sample pumpsare operated so that blood and diluent are simultaneously dischargedinto the mixing chamber. As indicated by the arrows in FIG. 4, bloodfollows the path represented by arrow 65 and diluent the pathrepresented by arrows 67. The two flows merge in the upper portion 68 ofthe mixing chamber, pass through axial passages 72 and into the lowerportion 69 of the mixing chamber and then through the restricted annularorifice into tip duct 64'.

Probe 15, with the structure just described shown schematically, isillustrated in FIG. 5 in conjunction with its pumps and associatedcontrols. As indicated, push button 35 is coupled to an autodilutercontrol block 70 with a voltage therefrom supplied back to LED displayelement 58. Control block 70, described in greater detail hereinbelow,upon receipt of a signal from push button 35 issues a reverse signal online 71 to the pump motor driver 73. This causes a voltage to besupplied to a small, e.g., 24 volt d.c., reversible motor 75 having onits shaft a gear 76 meshing with grears 77 and 78. Gear 77 has the samenumber of teeth as gear 76, e.g., 27 teeth, and gear 78 twice thatnumber. Gear 77 is fixed on the shaft of blood sample pump 79 and gear78 coupled to a diluent pump 81 through a one-way clutch 83. The one-wayclutch is oriented to permit diluent pump 81 to run only in the forwarddirection; nevertheless, both pumps are reversible in operation and, inorder to achieve the high precision volume displacement required,peristaltic-type pumps are preferred. Accordingly, while both pumps haveinlet and outlet flow passages or ports, the function of the flowpassages of the sample pump depends on the direction of operation, thus,the passage functioning as a discharge when the sample pump is runningforwardly, becomes an intake passage for aspiration of blood when thepump is reversed.

Both pumps have coupled to their forward-running inlet side tubes 80,80' which lead to the diluent supply 19. The forward running outputs ofthe sample and diluent pumps 79 and 81 are coupled through tubing 82,82' to sample and diluent conduits, respectively, of probe 35. Whenmotor 75 is operating in reverse, pump 79 runs in reverse to aspirateblood in the manner described above. When the probe is positioned overthe substrate, push button 35 is operated again and auto-diluter controlsystem 70 causes motor driver 73 to dispense the sample. This involves amomentary excitation of motor 75 in the reverse direction so that all ofthe blood specimen in the mixing chamber is drawn completely intoconduit 62 and then in the forward direction causing, because of thedifferent gear ratio, two parts of blood to be mixed with one part ofdiluent and dispensed by the probe.

The operation of motor 75 is controlled as will be described presentlyso that only the desired volume of diluted blood is deposited on thesubstrate. In the preferred embodiment, approximately 190 microliters ofsample are drawn in. When the button is pushed a second time, thereverse drive is energized briefly as explained above, e.g.,approximately 80 milliseconds, to clear the mixing chamber. After a420-second delay, motor 75 is energized in the forward direction todispense approximately 125 microliters of blood and 62.5 microliters ofdiluent for an approximate total quantity of 187.5 microliters of themixture being deposited on the substrate. Thereafter, probe 15 is placedin its holder.

FIG. 6 is a block-logic diagram of the control circuit 70 of FIG. 5including, for ease of reference, a diagrammatic representation of probe15 and its push button 35 and LED indicator 58. Also included are pumps79 and 81 with their drive motor 75 and the motor driver 73. Theremainder of the elements, which are enclosed within dashed lines,comprise control system 70 which is made up of logic circuit buildingblocks such as Texas Instruments' 7400 and 9300 series units. The logicis not shown in complete detail since the design will be evident tothose skilled in the art.

The basic elements include a start logic block 91, a cycle flagflip-flop 93, a wait flip-flop 95, a subcycle register 97, a cycleregister 98, a programmable timer 99 and delay flag 101. The output fromthe various modules is coupled through a number of gates. The forwardand reverse inputs into pump motor driver 73 is through AND gates 103and 105, respectively. These obtain an enabling input from an AND gate107 and obtain their second inputs respectively from OR gates 109 and111.

Programmable timer 99 can be preset with time settings as indicated bythe inputs thereto. As noted above, the probe first aspirates a bloodsample and thereafter continues aspiration to draw blood out of themixing chamber. The probe then dispenses the diluted specimen and, afterbeing placed back in its holder, is cleansed. The time of occurrence andduration of each of these steps (e.g., the times mentioned foraspirating and dispensing) are set into the programmable timer 99.

Upon the first depression of the button 35, inputs are provided to thestart logic 91 and to the flip-flops 93 and 95. The start logic providesan output to the cycle register 98 resetting it to the "aspirate 1"position. Flip-flops 93 and 95 are set and provide inputs to subcycleregister 97. The output flip-flop of 95 is also coupled to the LEDdisplay 58 to indicate to the operator that a cycle is in progress.Subcycle register 97 provides an output to the programmable timercausing it to generate an output representing the desired time for thefirst aspiration. Register 97 also provides an output on the linelabeled "Y" to the AND gate 107. At this point, delay flag flip-flop 101is reset so its Q output is a 1. AND gate 107 is now enabled and willhave a 1 output which is provided to AND gates 103 and 105. Since cycleregister 98 is in the "aspirate 1" position, there will be a 1 at theinput to OR gate 109 and thus a 1 at its output. As a result, AND gate103 has two 1 inputs and will provide a 1 output to pump motor driver 73causing motor 75 to run in reverse.

At the end of the "time-out" signal from programmable timer 99,flip-flop 101 is reset, disabling AND gate 107 which will, in turn,disable AND gate 103 stopping the motor. At the same time, an outputwill be provided resetting cycle flag flip-flop 93. Subcycle register 97will provide a shift output to shift cycle register 98 to "aspirate 2."Once the operator has positioned the probe over the substrate and againdepresses button 35, the cycle flag will again be set causing thesubcycle register to cause the programmable timer to output the time for"aspirate 2." In the manner indicated above, this aspiration will becarried out, the OR gate 109 now obtaining its input from "aspirate 2."At the end of that time, subcycle register 97 will provide a shiftoutput to cycle register 98 resulting in the "dispense" output (which isan input to OR gate 111). Programmable timer 99 will also be stepped tothe next output, i.e., the "dispense" output. Now, in the mannerdescribed above, AND gate 107 will be enabled for the preset timeenabling AND gates 103 and 105. For this step, there are no 1 inputs atOR gate 109 and thus, the motor will drive in reverse. However, one ofthe inputs to OR gate 111 is present so that AND gate 105 will have two1 inputs and will provide a forward signal to the driver. This willcause the motor to drive forward in the manner described above todeposit the diluted sample on the slide.

Thereafter, subcycle register 97 will shift cycle register 98 to the"clean" output simultaneously shifting programmable timer 99 to thatoutput. After a delay sufficient to permit the operator to place theprobe back in its holder, the "clean" cycle will be carried out in themanner described above with the pumps forcing diluent through bothdiluent and sample conduits of the probe.

At the end of this time, the "end cycle" signal from the subcycleregister along with the "end op" signal will be provided, resetting bothflip-flops 93 and 95. The LED display 58 will now indicate that a newcycle can be carried out. The outputs of flip-flops 93 and 95 are alsoprovided as enabling inputs to start logic 91 so that only whenflip-flops are reset, indicating a complete operating cycle has beenaccomplished, can the start logic become effective. In other words,pressing the button during a cycle will not have an effect (other thancausing a sample to be dispensed once aspiration has taken place).

As indicated above, once the diluted blood specimen has been placed onthe substrate, it is moved into the centrifuge chamber in enclosure 29(FIG. 1) where it is then spun.

As can be seen from FIGS. 7 and 8, carriage 27 for substrate holder 21slides into and out of enclosure 29 on a track 121 bolted to a base 123for the enclosure in any suitable and conventional fashion.

Within enclosure 29, above the area occupied by substrate holder 21during spinning, is a water-curtain forming assembly, comprising adeflector 125 and a spreader 127. A water conduit enters enclosure 29through an opening 129 and is joined to a coupling 131 passing throughdeflector 125 so as to feed water to the space between the deflector andspreader 127. The gap between the deflector and spreader is adjusted toabout 0.002 to 0.003 inches so that when water is supplied to the areatherebetween, a curtain of water forms around the periphery of thespinning substrate.

In order to permit insertion and removal of the substrate on holder 21,the assembly made up of the deflector 125 and spreader 127 is pivotedabout a point 133. A spring 135 biases the front of the assembly upwardto provide clearance for the substrate and holder 21 when retracted. Inthe fully inserted position shown, a cam 137 acts on a stop 139 securelymounted to a depending annular flange 141 of the spreader to push theassembly into the position shown against the force of the spring 135.Slide holder 21 is rotated to carry out the spinning by a motor 143secured to base 123 below the centrifuge chamber. The motor shaftprotrudes vertically upwardly through a carbon seal 145 into a springcoupling 147 slotted to engage with a complementary projection on theend of a shaft 149 on which slide holder 21 is supported. Shaft 149,journalled in a bearing 151 in carriage member 27, is actually a dual(coaxial) shaft assembly as will be explained in the ensuing descriptionrelating to FIG. 9.

To ensure that centrifuge motor 145 always stops with the notch ofcoupling 147 properly aligned, a detent disc 153 is attached to thedownwardly projecting lower shaft 155 of motor 143. Disc 153 contains adetent which is engaged by a detent mechanism 157 coupled to a solenoid159 through a coupling 161. Detent mechanism 157 is biased toward detentdisc 153 by means of a spring 163. When the centrifuge is spinning,solenoid 159 is energized to move the detent 157 away from the disc topermit free rotation. However, as the motor speed is slowed aftercompletion of the spin cycle, solenoid 159 is de-energized so that thedetent will snap into the notch of detent disc 153 and stop the motor atthe proper position to permit insertion and retraction of carriagemember 27 with the slide holder 21 thereon. Also provided is amicroswitch (not shown) which is closed when the detent is in place toprovide an indication that the motor is stopped so that power can beremoved to stop rotation.

Locking of carriage member 27 in place within the centrifuge chamber isaccomplished by a latch 167 operated by a solenoid 169 controlled by amicroswitch 171. When handle 25 is pushed in, a flange 173 thereoncloses microswitch 171 to de-energize solenoid 169 causing the latch 167to move upwardly and thus prevent the handle from being pulled out untilthe end of the spinning cycle when, in a manner to be describedpresently, the solenoid will again be energized.

Substrate holder 21 and its shaft 149 are shown in greater detail inFIG. 9. At one end (right-hand as viewed in FIG. 9) of the substrateholder, is a fixed, notched substrate gripping member 175 for engagementof an edge of a substrate; at the opposite end of the holder is amovable gripping member 177 adapted to engage on the opposite end of thesubstrate. Movable member 177 can slide toward and away from fixedmember 175 and is biased toward the fixed member by a spring 179 tosecure a substrate on the holder. Motion away from the fixed member canoccur, however, only when a detent lever 181 is pivoted about a pivotpin 183 so as to be clear of a portion 189 of slidable member 177.Detent lever 181 is pivoted by means of an inner shaft 191 coaxiallyslidably disposed within shaft 149 and having a portion 193 projectingfrom the lower end thereof. Detent lever 181 is biased into the engagingposition shown on the drawing by means of a spring 195. Projecting end193 is also shown on FIG. 7 and it can be seen from both FIGS. 7 and 9that, by pushing upward on projecting end 193, lever 181 will beangularly displaced to permit movement of member 177 toward and awayfrom fixed member 175.

When the substrate holder assembly is within the centrifuge chamber, asshown in FIG. 7, the detent mechanism is in the position shown in FIG. 9and, as movement of member 177 is not possible, the substrate is heldfirmly during spinning. However, as handle 25 is pulled out to withdrawthe slide holder as carriage member 27 nears the end of its travel, theprojecting portion 193 of shaft 191 rides up on a ramp 197 attached totrack 121 with the carriage in fully withdrawn position, shaft 191 ispushed upward causing the opposite end of lever 181 to be moved downwardso that the substrate can be removed and a new substrate inserted bydisplacing gripping member 177 outwardly against the pressure of spring179.

FIG. 10 is a plumbing schematic illustrating the manner in which wateris provided for the water curtain around the circumference of thespinning slide and also showing the manner in which the external portionof probe 15 is washed. Deflector assembly 125, 127 is schematicallyrepresented in FIG. 10, as is a bowl 201, which serves as a catch basisfor water from the curtain. Bowl 201 is formed with a depression 203(shown in FIG. 7) which connects via a coupling 205 to a drain orexhaust line 224. Also schematically shown in FIG. 10 is the previouslymentioned holder 207 for probe 15 which includes two wash rings 209 and211. Water from a spigot at line pressure is routed through a pressureregulating valve 219, a solenoid valve 221, and then to a flow valve223. Typically, pressure regulator 219 is set for 50 psi. Water fromflow valve 223 enters deflector assembly 125, 127 through inlet 131,details of which are shown in FIG. 17, and passes through to form thecurtain of water around the spinning slide. Water is also suppliedthrough a flow valve 229 to the wash rings 211 and 209 at a flow rateset so that the water level never rises about the upper wash ring 211.Wash water for the external part of the probe, along with diluentflushed through the probe in the manner described above, flows outthrough a drain 231 in the bottom of probe holder 207 via a drain line233. Drain lines 224 and 233 are connected to an exhaust pump 225 drivenby a motor 227. As indicated above, in connection with FIG. 2, exhaustpump 225 continues to run after the solenoid valve for the watersupplying curtain is turned off. Inasmuch as the centrifuge chamber issealed by movement of the slide holder carriage into the spin position,forced drainage creates a partial vacuum within the chamber to draw offresidual aerosol before it is opened by retraction of the spinnerassembly.

FIG. 11, a basic block-logic diagram of the control system for thespinner, is essentially an implementation of the flow diagram of FIG. 2utilizing standard logic-building blocks similar to those used for theprobe control.

Operation of the system requires appropriate signal inputs to a startlogic network 241. In addition to a signal from the operator on line243, start logic 241 receives inputs from a logic block 245 indicatingthe status of parameters determining whether or not the system is readyto start, e.g., angular position of the spinner, position of thecarriage. To this end, block 245 receives a signal from a "power-on"reset module 247 and, via line 249, an indication of the substrateholder position from the microswitch of FIG. 7. When starting initially,it is important that the substrate holder be properly aligned so thatthe carriage can be retracted from the centrifuge chamber. Thus,depending on these inputs, a command is given either to initiateposition, i.e., to position the centrifuge motor 143 of FIG. 7 so thatthe coupling notch is properly aligned or to set the ready light if thathas already been done. These outputs are provided from logic block 245,which will contain necessary logic gates for the purpose, to start logicblock 241.

Then, when receiving an input from the operator on line 243, start logic241 provides a number of control signal outputs through an input/outputblock 251: (1) on line 253, a signal to engage the position relay orsolenoid 159; (2) on line 255, a signal to de-energize carriage lockingsolenoid 169; and (3 ) on lines 257 and 258, signals to control theready lamp and purge lamp indicating whether or not a cycle is inprogress or ready to start. In the condition just mentioned, where thecycle has been started, the ready lamp will, of course, be extinguished.Start logic 241 also provides a signal to a subcycle register 261, whichoperates much in the manner of the subcycle register (97, FIG. 6) of theprobe control system to carry out the various subcycles indicated inFIG. 2. The output from subcycle register 261 is provided to a cycleregister 263 which provides four different outputs designated I, II, IIIand IV:

I. The first output, which corresponds to block 43 of FIG. 2, providesan input to a pump motor driver 73, to a driver 265 for the drain pumpmotor 227 of FIG. 10, and to the water solenoid valve 221.

II. The register then steps to the next function, which corresponds toblock 45 of FIG. 2, to carry out the spin program. This output isprovided to a spin motor control block 267 which includes conventionalintegrating means for accelerating and decelerating the motor. The motoris accelerated to a speed of approximately 35 rpm, maintained at thatspeed for a short time, and decelerated.

III. Once the spin cycle is carried out, register 263 steps to the"pump-off" output, represented by block 47 on FIG. 2. This output isutilized directly to turn off the water solenoid and, through a delay269, to terminate the exhaust pump drive to get the four second delaymentioned above.

IV. In the last register step, the position cycle is carried out inwhich an output to de-energize the solenoid 159 so that the centrifugemotor, which is now rotating slowly will be stopped in the requisiteposition to enable retraction of substrate carriage.

During the various operations I, II, III and IV, indications are fedback to a "busy decode" module 271 which is coupled through an AND gate273 to three flip-flops 275, 277 and 279 which are used to step subcycleregister 261 and concomitantly, cycle register 263 through the varioussteps. As the inputs to the busy decode module correspond to the outputsof cycle register 263, in each case at the beginning of a particularstep the start flip-flop is set to advance the register 263. As long asthis step is being carried out, an output from busy decode 271 preventsstarting another cycle. However, once the cycle is completed, the busyflip-flop is reset and the program can continue in accordance with anoutput from the continue flip-flop. This ensures that a subsequent stepis not started while a previous step is in progress.

Thus, an improved spinner for preparing blood smears on slides has beenshown. Although specific embodiments have been illustrated anddescribed, it will be obvious to those skilled in the art that variousmodifications may be made without departing from the spirit of theinvention, which is intended to be limited solely by appended claims.

What is claimed is:
 1. A method of preparing a blood film on a planar substrate comprising the steps of:depositing a blood specimen on a substrate; spinning the substrate at a high angular rate about an axis perpendicular to its plane; and surrounding the substrate with a flowing curtain of liquid positioned to intercept and collect blood particles and aerosols spun off the substrate.
 2. A method according to claim 1 including the further step of continuously directing the liquid from said curtain into a waste line during the spinning of said substrate.
 3. A method according to claim 2 wherein the spinning is carried on in an enclosed chamber from which said liquid is pumped and including the further step of terminating the liquid curtain flow while continuing to pump the liquid from the enclosed chamber to form a partial vacuum therein tending to remove any residual blood in aerosol form.
 4. A method according to claim 3 wherein the blood specimen is diluted before being deposited on the substrate.
 5. A method according to claim 4 wherein dilution is in a ratio of 2 parts blood to 1 part diluent and the diluent is physiological saline solution.
 6. A method according to claim 5 wherein the substrate is spun at a speed in the range of 3000 and 4000 rpm.
 7. A method according to claim 6 wherein the substrate is spun at 3500 rpm.
 8. A method according to claim 4 wherein said dilution is effected by steps comprising:a. drawing a blood specimen into an aspirating probe; and b. discharging said specimen in a measured amount along with a measured amount of diluent from said probe.
 9. A method according to claim 8 including the further step of mixing said specimen and diluent in the tip of said probe before discharging. 